RO134197A2 - Process for preparing composite hydrogels based on collagen and silver nanoparticles for preventing wound infections - Google Patents

Abstract

The invention relates to a process for preparing composite hydrogels based on collagen and silver nanoparticles for preventing wound infections. According to the invention, the process consists in directly reducing Ag ions, in situ, in the collagen gel, in the presence of reducing agents, such as NaBH, citric acid, followed by hydrogen reticulation in the presence of a reticulation agent such as, glutaraldehyde, to result in a collagen matrix with a content of Ag nanoparticles within the range 0.1...5%, having an antimicrobial activity against adhered and in-suspension Gram-positive and Gram-negative bacteria and immunomodulating activity.

Description

PATENT DESCRIPTION: PROCESS FOR OBTAINING COLAGEN-BASED COMPOUND HYDROGELS AND SILVER NANOPARTICLES FOR THE PREVENTION OF WOUND INFECTIONS

Skin wound healing is a dynamic and complex process involving the extracellular matrix, soluble mediators and the activation of various cell types. in a normal healing process, there is a specific transition from the inflammatory phase to tissue regeneration followed by angiogenesis and tissue reorganization (Dovi JV, Szpaderska AM 2004). The healing process is slowed by the existence of a prolonged inflammatory phase and necrotic tissue that promotes bacterial adhesion and proliferation, as well as the formation of biofilms (Zhao et al., 2013). Microbial biofilms are present in approximately 6% of acute injuries and over 90% of chronic injuries (Attinger and Wolcott, 2012). In general, infections with biofilm production have a slow progression but, once established, are difficult to combat (Lazăr et al., 2010).

The management of skin wounds lies in the use of local treatments in order to prevent / treat wound infections and fast and effective tissue regeneration. Wet and dry collagen structures are currently considered one of the most effective strategies in the treatment of acute and chronic skin wounds (Grigore et al., 2017).

Inorganic nanoparticles have numerous applications in the biomedical field and in the pharmaceutical industry. Certain types, such as metallic, silver (Ag), copper (Cu), titanium (Ti), iron (Fe) and zinc (Zn), have demonstrated significant antimicrobial activity and are currently being investigated for the development of new antimicrobial strategies, as alternatives to the use of antibiotics. It is well known that silver used in various forms (eg elemental form, inorganic compounds or organic or inorganic formulations) may have a pronounced antimicrobial effect and even promote wound healing when included in various dressings (Michaels et al., 2009).

The problem that the invention solves is the inhibition of microbial biofilms, one of the main causes of delayed healing of chronic wounds of patients with other underlying conditions (e.g., diabetes, circulatory disorders, etc.). Chronic wounds are a favorable niche for the development of biofilms due to the altered immune response that increases susceptibility to infections, while the necrotic tissue that appears locally promotes microbial adhesion. Biofilms formed at SANIMED University from the University Institute of Virology

INTERNATIONAL Bucharest Politehnica Ștefan S. Nicolau, Bucharest

IMPEX SRL Bucharest

Catal in-Robertino

Hideg

Prof. Dr. Mircea

Dumitru

Prof. Mihnea

Costoiu

Dr. Carmen Cristina

DIACONU 2018 01009

29/11/2018 the level of chronic wounds influences the functioning of the cells involved in wound healing (neutrophils, endothelial cells, fibroblasts), skin immune response and angiogenesis (Mihai et al., 2018). Most collagen-based hydrogels or dressings available on the international market are collagen products that are used with caution in the treatment of wounds, are usually handled by trained medical personnel and, although they have many advantages in healing skin wounds, most do not have antimicrobial effect.

The solution proposed by the present invention is the development of advanced (multi) functional hydrogels, based on collagen and inorganic nanoparticles with antimicrobial effect, which facilitate the healing of acute and chronic skin wounds even in the case of associated infections. The new products could benefit the elderly, paraplegics or the immobilized due to other causes, chronic diseases (i.e. diabetes), but also active people with various chronic wounds, burns, chemical skin diseases, surface or deep traumas and mechanical traumas.

Advantages of the invention:

Collagen-based biomaterials and silver nanoparticles have the advantage of promoting wound healing by:

Providing a skin precursor;

maintaining optimal humidity and aeration parameters;

biocompatibility;

hemostatic properties;

preventing the formation of microbial biofilms.

The following are examples of the invention in connection with:

Fig. 1. How to obtain collagenous materials with Ag nanoparticles (in one or two steps).

Table 1. Technical description of the collagenous materials obtained.

Fig. 2. Morphology of HeLa cells developed on the surface, at the area of contact and away from collagenous material

Fig. 3. Quantification of cell viability in the presence of collagenous materials with Ag.

SANIMED INTERNATIONAL IMPEX SRL The University of Bucharest Polytechnic University of Bucharest Ștefan S. Nicolau Institute of Virology, Bucharest Catalin-Robertino Prof. Dr. Mircea Prof. Mihnea Dr. Carmen Cristina Hideg Dumitru Costoiu Diaconu

and 2018 01009

29/11/2018 / 0?

Fig. 4. Expression of markers inducing apoptosis by extrinsic mechanisms in the presence of collagenous materials with Ag.

Fig. 5. Expression of markers activating cellular metabolism in the presence of collagenous materials with Ag.

Fig. 6. Overlapping histograms of the cell cycle of HeLa cells maintained in the presence of collagenous materials with Ag.

Fig. 7. Dot blot representation of THP-1 cell activation in the presence of collagenous materials (hCLAT -human cell line activation test)

Fig. 8. Expression of inflammatory cytokines in epithelial cell cultures in the presence of collagenous materials with Ag.

Fig. 9. Graphical representation of the antimicrobial effect of collagenous materials quantified by the qualitative method.

Fig. 10. Graphical representation of the antimicrobial effect of collagenous materials quantified by the quantitative method against planktonic microbial cells.

Fig. 11. Graphical representation of the antimicrobial effect of collagenous materials quantified by the quantitative method against adherent microbial cells.

Fig. 12. Preparation of the animal, biopsy excision of a portion of the skin, and implantation of collagenous material.

Fig. 13. Appearance of the wound 4 days after implantation of the collagenous material.

Fig. 14. Appearance of the wound 4 days after bacterial inoculation and implantation of collagenous material.

Fig. 15. Analysis of IL-1 expression in the wound

Fig. 16. Histopathological analysis of the wound level

EXAMPLE 1. OBTAINING COMPOUND HYDROGELS BASED ON

COLLAGEN AND SILVER NANOPARTICLES j

Collagen was obtained by solubilization in an acid medium, in the presence of the proteolytic enzyme pepsin. Implementation methodologies were carried out in dedicated work areas for each operation, with controlled environmental and hygiene conditions. In order to obtain the collagen material, rich sources of collagen were exploited, respectively raw materials of bovine or equine origin, of controlled origin from a sanitary-veterinary point of view. The raw material carefully selected and with registered traceability, was subjected to the process of manual primary processing (preliminary technological stage, which involves a succession of operations, respectively thawing

SANIMED INTERNATIONAL IMPEX SRL The University of Bucharest Polytechnic University of Bucharest Ștefan S. Nicolau Institute of Virology, Bucharest Catalin-Robertino Prof. Dr. Mircea Prof. Mihnea Dr. Carmen Cristina Hideg Dumitru Costoiu Diaconu

and 2018 01009

29/11/2018 controlled, decontamination, removal of non-collagenous or lipid parts, washing with at least five volumes of pure water, related to the processed raw material), but also a secondary processing (controlled technological stage, which involves crushing the raw material, using a industrial shredder type equipment, with Simian sieve, with a mesh diameter of 3-5 mm). The raw material thus processed was portioned, in the form of ready-to-use packages and subjected to rapid freezing at -20 ° C. The rules for interphase control specific to raw materials of animal origin were complied with, and the batches of raw materials used were entered into the database on the extended traceability of the product.

The raw material of bovine origin was subjected to an enzymatic treatment process in an acid medium in order to solubilize the collagen. An industrial homogenizer type equipment was used with interchangeable tanks with a useful capacity of 50 L, which ensures a good homogeneity in reaction mass, with controlled rotation speed (minimum 40 rotations / minute). The collagen extraction mixture involved an acidic environment (pH 2-3), provided by a solution of 0.5M acetic acid (20L), preteolytic enzyme, pepsin the ratio between pepsin and dry matter of the tissue used, being 1 : 99-1: 5, such as 1: 9. Collagen solubilization was performed for a period of 48 hours, under temperature controlled conditions (20-25 ° C), with monitored intermittent agitation (40 rotations / minute, for 20 minutes every hour). Removal of undigested tissue was performed by centrifugation (4 ° C, 9000 rpm, for 15 minutes), obtaining a collagen gel, with a pH value of 2.3-2.5, with a whitish-translucent appearance, with a viscous consistency. Collagen purification was performed by dialysis, against pure water, or by diafiltration, using ceramic columns (with an equivalent porosity of 20 nm), countercurrent to the collagen gel circuit, until the pH value was adjusted in the range of 6.5-7. .

Collagen-based composite hydrogels and Ag nanoparticles were obtained by reducing Ag ⁺ to Ag °, in-situ (by adding reagents to the collagen hydrogel), hereinafter referred to as "in one step", respectively obtaining metal nanoparticles in advance, and the subsequent addition of collagen hydrogel, later called "two-step". in both cases the samples were subjected to vigorous mechanical homogenization, in order to obtain a more uniform distribution of the particles in the polymer mass. In this respect, NaBEL was used as a reducing agent,

SANIMED University of

INTERNATIONAL Bucharest

IMPEX SRL

University Institute of Virology

Ștefan S. Nicolau Polytechnic, Bucharest

Bucharest

Catalin-Robertino

Hideg

Prof. Dr. Mircea

Dumitru

Prof. Mihnea

Costoiu

Dr. Carmen Cristina

DIACONU 2018 01009

29/11/2018 respectively a reducing mixture based on the synergistic effect of NaBH4 and citric acid (biocompatible reducing agent, of the antioxidant type, but with a reducing effect known to be lower than that of NaBEL). Citrate ions also play other roles in the process of reducing silver ions, such as stabilizing and complexing agent. In order to control the morphology of the obtained metal particles and to maintain them in dispersed form, in the synthesis process PVP (polyvinylpyrrolidone) was used, which favors the obtaining of platelet, triangular structures with chamfered corners. A dilute solution of glutaraldehyde was used for cold crosslinking of the collagen for at least 24 hours. The resulting composites are summarized in Table 1.

To obtain all the composite hydrogels, a dilute aqueous solution of ⁴ M AgOCl and ¹ M sodium borohydride (NaBEU) was used as precursor. In the case of samples with mixed reducing agent and dispersing agent, 0.3M citric acid was used, respectively polyvinylpyrrolidone (PVP) 0.007 M. finally 5 mL of 30% hydrogen peroxide was added and stirring was maintained for about 10 minutes until a slightly dark blue color was obtained (color due to the size of the nanoparticles).

In order to analyze the degree of release of nanoparticles from the formation of hydrogels, inductively coupled plasma mass spectrometry (ICP-MS) was used. This method of elemental analysis is indicated if it is desired to analyze small quantities of »>

sample, in which the concentration of the analyte is low (of the order of ppt or ppb). To this end, solutions of the same concentration of collagen and Ag-based hydrogels were made in phosphate buffered saline from which samples were collected at fixed time intervals and analyzed using ICP-MS. The results obtained in the case of Ag samples show a slow release of the nanoparticles from the hydrogel, the silver concentration in the PBS solution being ~ 28pg / L after 120h from immersion. This aspect demonstrates that the materials obtained can provide prolonged antimicrobial protection, 5-7 days, without the need for replacement, if used as dressings.

Figure 1 shows the technological flow scheme for obtaining composite hydrogels based on collagen and silver nanoparticles (in one stage, respectively in two stages).

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The University of Bucharest

Prof. Dr. Mircea

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University Institute of Virology

Ștefan S. Nicolau Polytechnic, Bucharest

Bucharest

Prof. Mihnea Dr. Carmen Cristina

COSTOIU DIACONU a 2018 01009

11/29/2018

EXAMPLE 2. QUALITATIVE AND QUANTITATIVE ANALYSIS OF TOXICITY OF AG COLAGENIC MATERIALS AND INTIMATE MECHANISMS OF INTERACTION WITH MAMMAL CELLS 5

Adherent cell line HeLa (American Cell Type Collection [ATCC] CCL-2) was used for quantitative analysis of Ag-induced collagen hydrogel toxicity. Samples of collagenous materials with Ag (2cm ² ) previously sterilized by UV irradiation were distributed in 6-well plates, after which they were washed with sterile saline and equilibrated in medium, stabilizing the pH value at 7.2-7, 4. Subsequently, 3x10 ⁶ cells were added to each well containing a fragment of collagenous Ag materials. The plates were incubated in modified Dulbecco's medium (DMEM: F12) (Sigma, USA), supplemented with thermally inactivated 10% fetal bovine serum (Sigma, USA), at 37 ° C, in a humid atmosphere with 5% CO2 for 24 ° C. for hours. The cell morphology was observed under the Zeiss Observer Dl microscope and the cells were photographed in phase contrast using the AxioCam MRm camera.

Cells developed at the point of contact with Ag collagenous materials show a specific morphology of dead, apoptotic cells (Figure 2). Subsequently, the collagenous Ag materials were removed from the culture plates, fixed in alcohol and stained with propidium iodide. Subsequently, both sides of each product were examined under a fluorescence microscope, confirming that HeLa cells did not adhere to collagenous materials.

The quantification of toxicity induced by collagenous materials with Ag was performed using the trypan blue test and the results are presented as% viability compared to the control in figure 3. The viability of HeLa cells was> 60% for all tested materials, and> 80% for AgNCh, collagen + NaBH4 in one step and AgNCU, NaBH4, citric acid, PVP + collagen in two steps, a percentage that, according to biomaterial cytotoxicity assessment standards, indicates a low level of cytotoxicity.

To evaluate the mechanisms of toxicity induction, cells maintained for 24 h in the presence of Ag-based composite hydrogels were trypsinized, centrifuged, and subjected to RNA extraction with Trizol (Thermo Fisher Scientific, USA), according to the manufacturer's protocol.

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and 2018 01009

29/11/2018 / Of

The extracted RNA was dried on ice and resuspended in 20 μL of nuclease-free water. RNA concentration and purity were determined using the Eppendorf BioPhotometer plus spectrophotometer (Eppendorf, Germany). Total RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit with RNAse inhibitor (Thermo Fisher Scientific, USA) starting from 2 pg RNA. The reverse-transcription reaction was performed in the BioRad DNA ENGINE Dyad thermocycler, using the program: 25 ° C, 10 min .; 37 ° C, 120 min .; 85 ° C, 5 min. 50 ng cDNA were introduced into the qPCR reaction for each sample, the amplification mix also containing the reaction mix, TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, USA), and primers, Taqman Gene Expression Assays (Thermo Fisher Scientific, USA). ) as follows: Casp7 (HsOOl 69152_ml), Casp8 (HsOOl 54256_ml), Casp9 (Hs00154261_ml), BAX (HsOOl 80269_ml), BCL2 (Hs00153350_ml), MCL1 (HsOOl 72036_ml), HR0051) and Expression level quantification was performed on the StepOne Plus Real-Time PCR System (Thermo Fisher Scientific, USA), each experiment being performed in triplicate. The conditions of the amplification reaction were: 95 ° C, 10 min .; 40 x (95 ° C, 15 sec .; 60 ° C, 1 min.). The results were analyzed with StepOne Software v2.3 (Thermo Fisher Scientific, USA), comparing the relative expression levels being performed by the AACt method. The expression level of the GAPDH gene was used as endogenous control.

Apoptosis can be initiated by one of two separate pathways (intrinsic or extrinsic pathway), both leading to the final, effector, execution phase. In the presence of collagenous materials, the intrinsic pathway, triggered by internal stimuli (biochemical stress, DNA alteration, starvation, etc.) is inhibited by reducing gene expression for caspase 9 and Bcl2 and Bax proteins. Instead, the extrinsic pathway, triggered by ligand binding to dead receptors on the cell surface (TNFR1 / FAS) is activated by Ag-doped collagen hydrogels, initiating the activation of downstream caspases, respectively caspase 8 and caspase 7 (figure 4). .

Beta-arrestin 1 (ARRB1) is an adapter protein with a wide range of cellular and molecular functions, including in the regulation of cellular hypoxia by co-localization and physical interaction with HIFla, which facilitates glucose absorption and glycolysis. Modulation of ARB1 expression under the influence of collagenous materials demonstrates interference with cellular metabolism (Figure 5). So,

SANIMED INTERNATIONAL IMPEX SRL The University of Bucharest Polytechnic University of Bucharest Ștefan S. Nicolau Institute of Virology, Bucharest Catalin-Robertino Prof. Dr. Mircea Prof. Mihnea Dr. Carmen Cristina Hideg Dumitru Costoiu Diaconu

and 2018 01009

29/11/2018 the materials AgNCh, collagen, citric acid, PVP, NaBH4 and, respectively, AgNCL, NaBH4, collagen induced the significant decrease of this protein, while AgNCh, collagen + NaBFL in one stage and AgNCh, NaBH4, acid citrus, PVP + collagen in two stages induced the increase of the expression of this protein.

TRPC1 (Transient receptor potential channel 1) modulates intracellular Ca2 + concentrations, controlling critical cytosolic and nuclear events. A typical feature of TRP channels is their ability to be activated by a wide range of external stimuli (including light, sound, chemicals, temperature and touch), as well as changes in the local environment (Venkatachalam and Montell, 2007, Nilius and Owsianik, 2011). TRPC1 is an important component of the hypoxia response. Increased expression of CPR facilitates the influx of Ca, a phenomenon facilitated by hypoxia. Thus, the drastic decrease of TRPC1 expression in the presence of collagenous materials with Ag, supports the hypothesis that they are not inducers of hypoxia, and do not facilitate the influx of calcium (figure 5).

EXAMPLE 3. ANALYSIS OF THE INFLUENCE OF AG COLAGENIC MATERIALS ON THE CELL CYCLE OF HELA CELLS

HeLa cells maintained for 24 h in the presence of Ag-based composite hydrogels were trypsinized, washed in phosphate buffered saline (TFS) and fixed in 1 ml of 70% cold ethanol for at least 30 minutes at -20 ° C. Subsequently, the cells were washed in TFS, resuspended in 100 μΐ TFS, treated with RNAase (final concentration 1 mg / ml) at 37 ° C for 30 minutes and stained with PI (final concentration 100 pg / mL) for 30 minutes. minutes at 37 ° C, with regular stirring. The samples were read on the Beckman Coulter XLM flow cytometer and analyzed with the FlowJo program.

Cell cycle analysis showed that the hydrochloride composites did not interfere with the cell cycle phases of HeLa cells maintained in the presence of materials (Figure 6).

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Catalin-Robertino

Hideg

The University of Bucharest

Prof. Dr. Mircea

Dumitru

Polytechnic University of Bucharest

Prof. Mihnea

Costoiu

Ștefan S. Nicolau Institute of Virology, Bucharest

Dr. Carmen Cristina

Diaconu

29/11/2018 j _n / £>

and 2018 01009

EXAMPLE 4. ANALYSIS OF THE INFLUENCE OF AG COLAGENIC MATERIALS ON CELL ACTIVATION (H-CLAT -HUMAN CELL LINE ACTIVATION TEST)

In this example, the THP-1 cell line [ATTC TIB-202] (a human monocytic leukemia cell line) was used as a surrogate for dermal dendritic cells. This test method uses surrogate cells to assess the migration of skin dendritic cells to adjacent lymph nodes. A skin sensitization event leads to increased CD54 and / or CD86 expression that is used to assess cell activation (Ashikaga et al., 2010; Reisinger et al., 2015; Galbiati et al., 2016).

Thus, 4x10 ⁵ THP-1 cells were seeded in 24-well plates in RPMI medium supplemented with 10% fetal bovine serum. To these was added the sample of collagenous material with Ag (1 cm ² ). At 24 hours, the collagenous material was removed and the cells were centrifuged, labeled with CD86 (B72) -PC5.5 (Beckman Coulter B30647) and anti HLA-DR-Pacific Blue (Beckman Coulter A74781). The labeled cells were analyzed on the Astrios Beckman Coulter MoFLO flow cytometer using 488-710 / 45 Height log (CD86-PC5.5) and 405 - 448/59 Height log (anti HLA-DR-Pacific Blue). using Kaluza Analysis software version 1.3.

After removal of the tested collagen material, CD86 and HLA-DR expression was quantified by flow cytometry. The relative value of fluorescence compared to the control (cells maintained without collagenous material) is shown in Figure 7. The results demonstrate that Ag collagenous materials do not induce the activation of THP-1 cells.

EXAMPLE 5. INFLUENCE OF AG COLLAGEN MATERIALS ON THE EXPRESSION OF INFLAMMATORY CYTOKINES IN EPITHELIAL CELL CULTURES

Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, USA) and reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit with RNAse inhibitor (Thermo Fisher Scientific, USA) according to previous protocols. The qPCR reaction occurred

SANIMED The University of University Institute of Virology INTERNATIONAL Bucharest Polytechnic Ștefan S. Nicolau, Bucharest IMPEX SRL Bucharest Catalin-Robertino Prof. Dr. Mircea Prof. Mihnea Dr. Carmen Cristina HEDEG Dumitru Costoiu Diaconu

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29/11/2018 j made using the TaqMan Gene Expression Master Mix kit (Thermo Fisher Scientific, USA), and primers, Taqman Gene Expression Assays (IL1B (HsOl555410_ml), IL6 (Hs00174131_ml), IL8 (Hs00174103_ml).

The results shown in Figure 8 show that Ag collagen hydrogels induce an increase in the expression of pro-inflammatory cytokines IL6 and IL-8, but a decrease in IL-1 expression. It is well known that inflammation (also called the lag phase of healing an ulcer) is an essential step in wound healing. This phase is mediated by leukocytes, which, with the help of reactive oxygen radicals with bactericidal properties and released proteases, remove by enzymatic digestion dead tissue cells and bacterial cells; inflammation lasts for several days, ending with apoptosis of inflammatory cells (Gilroy et al., 2004; Eming et al., 2007). IL-6 is an essential mediator of the acute phase response, following tissue injury and is characterized by fever, leukocytosis, increased vascular permeability. IL-8 is a chemokine with chemotactic effect against neutrophils, eosinophils and promotes the inflammatory response. Increases the density of adhesion molecules on the neutrophil membrane. IL-8 stimulates the activation of neutrophils (transendothelial migration, release of lysosomal enzymes, intensification of oxidative metabolism, generation of the superoxide anion in the inflammatory focus). It has effects on non-lymphoid cells: it is haptotactic (induces the migration of epithelial cells to the surface). Therefore, the increase in the level of these cytokines in the presence of collagenous materials that favor the influx of leukocytes at the site of skin lesion and the installation of the acute inflammatory phase. Decreased IL-1 levels can be considered a positive result, as various studies have indicated the correlation between the trend of chronic and delayed wound healing and high levels of IL-1 (Jiang et al., 2014).

EXAMPLE 6. QUALITATIVE ANALYSIS OF THE ANTIMICROBIAL EFFECT

To highlight the antimicrobial effect of collagen-based composite hydrogels and Ag nanoparticles, the following strains were used:

Pseudomonas aeruginosa (1 laboratory strain, P. aeruginosa ATCC 27853 and 1 clinical isolate, P. aeruginosa 202 = isolated from wound secretion, multidrug-resistant strain);

SANIMED INTERNATIONAL IMPEX SRL The University of Bucharest Polytechnic University of Bucharest Ștefan S. Nicolau Institute of Virology, Bucharest Catalin-Robertino Prof. Dr. Mircea Prof. Mihnea Dr. Carmen Cristina Hideg Dumitru Costoiu Diaconu

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29/11/2018 / &!

Staphylococcus aureus (1 laboratory strain, S. aureus ATCC 25923 and 1 clinically isolated,

S. aureus MRSA 2 = resistant to methicillin, isolated from wound secretion).

The strains were kept on nutritious broth with 20% glycerol at -80 ° C. For antimicrobial tests, the microorganisms were seeded on nutrient agar (bacterial strains). The colonies obtained were used to obtain suspensions in AFS (sterile physiological water) of optical density 0.5 Mc Farland (l-3xl0 ⁸ CFU / mL).

To perform the qualitative analysis of the antimicrobial effect, a method derived from the standardized antibiogram method (Kirby Bauer) was used. On an agar medium distributed in Petri dishes, the test microorganism is seeded in a cloth with the swab. Subsequently, the samples of dehydrated collagen dressings, of equal dimensions (diameter 6 mm and thickness of 3 mm), sterilized by UV exposure for 20 minutes on each side, are placed at equal distances (approx. 3 cm). To test the nanoparticle suspensions used in the production of nanoparticle-functionalized collagen hydrogels, 6 mm diameter sterile filter paper discs are added to the Petri dishes sown in cloth, over which 10 pL of the nanoparticle dispersions are added to the concentration used to obtain the hydrogel. (2% and 3%, respectively).

The Petri dishes are incubated for 18 hours at 37 ° C and then the diameters of the growth inhibition zones around the collagen disks containing different types of nanoparticles are analyzed. The results presented in figure 9 prove that the AgNCh NaBFU + collagen material shows the most pronounced antimicrobial effect for both S. aureus and P. aeruginosa strains.

EXAMPLE 7. QUANTITATIVE ANALYSIS OF THE ANTIMICROBIAL EFFECT

Inhibition of the growth of planktonic (floating) microorganisms in the presence of materials

To test the effect of the obtained materials on the growth of microorganisms in liquid medium (planktonic cultures), the obtained materials were cut in the form of discs with a diameter of 6 mm and a thickness of approx. 3mm and sterilized by exposure to UV radiation for 20

SANIMED INTERNATIONAL IMPEX SRL The University of Bucharest Polytechnic University of Bucharest Ștefan S. Nicolau Institute of Virology, Bucharest Catalin-Robertino Prof. Dr. Mircea Prof. Mihnea Dr. Carmen Cristina Hideg Dumitru Costoiu Diaconu

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11/29/2018 min on each side. A fragment of sterile material was deposited individually in a well of a sterile 24-well plate. Over the deposited materials, 1 mL of liquid medium (simple broth) was added to the wells and then 10 pL of 0.5 McFarland density microbial suspension, prepared in AFS (sterile physiological water, 0.9% NaCl soil). The 24-well plates thus prepared were incubated at 37 ° C for 24 h. After the expiration of the incubation time 200 pL of the obtained microbial suspensions were transferred to plates with 96 sterile wells and the turbidity of the microbial cultures (absorbance) was measured spectrophotometrically at 600 nm.

For the tested P. aeruginosa strains, obvious differences of inhibition of growth and multiplication of planktonic cultures belonging to the laboratory strain were observed (ATCC 27853), compared to the results obtained for the tested nosocomial strain. Thus, in the case of P. aeruginosa strain ATCC 27853, the increase was significantly lower compared to clinical strain 202, the differences being obvious especially in the case of the AgNCh PVP-Ac citric NaBFU + collagen sample.

in the case of the analyzed 5. aureus strains, an inhibition of growth and multiplication of suspended microbial cells can be observed in the presence of all types of collagen hydrogels containing bioactive Ag nanoparticles, differences being found in the inhibition of laboratory strain growth compared to nosocomial (figure 10).

Inhibition of adhesion and biofilm formation

To test the effect of collagenous materials on adhesion capacity and biofilm production, the obtained materials were cut in the form of discs with a diameter of 6 mm and a thickness of approx. 3mm and sterilized by exposure to UV radiation for 20 min on each side. A fragment of sterile material was deposited individually in a well of a sterile 24-well plate. Over the deposited materials, 1 mL of liquid medium (simple broth) was added to the wells and then 10 pL of 0.5 McFarland density microbial suspension, prepared in AFS (sterile physiological water, 0.9% NaCl soil). The well plates thus prepared were incubated at 37 ° C for 24 h. After incubation the materials were washed with AFS and the medium was changed to develop biofilms on their surface. The plates were incubated for 24 hours. After

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Catal in-Robertino

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The University of Bucharest

Prof. Dr. Mircea

Dumitru

Polytechnic University of Bucharest

Prof. Mihnea

Costoiu

Ștefan S. Nicolau Institute of Virology, Bucharest

Dr. Carmen Cristina

DIACONU 2018 01009

29/11/2018 / 2â At the end of the incubation period, the sample on which the biofilm was developed was washed with AFS and deposited in a sterile Eppendorf tube in one mL AFS. The tube was vortexed vigorously for 30 s and sonicated for 10 s to detach the cells from the biofilm. The cell suspension obtained was diluted and various dilutions were seeded on plates with solidified culture medium in order to obtain and quantify the number of colony forming units (CFU) / mL.

The analysis of the adhesion capacity and development of microbial biofilms on the surface of the obtained collagen samples shows that this phenotype is significantly reduced on the surface of dehydrated hydrogels containing Ag nanoparticles in different variants. In the case of both P. aeruginosa and S. aureus strains, the most pronounced antibiofilm effect was registered in the case of collagen materials and Ag nanoparticles obtained in two stages with NaBFU reducing agent (AgNCL + NaBFLi + collagen) (figure 11).

EXAMPLE: IN VIVO ANALYSIS OF THE ANTIBACTERIAL EFFICIENCY OF AG COLAGENIC MATERIALS

The excision wound model was used to study the repair processes and antibacterial efficiency of nanostructured materials. The experiments using white laboratory mice CD1 {Mus musculus) were approved by the Sanitary Veterinary and Food Safety Directorate (Authorization no. 310 of 31.10.2016 / NanoColaGel project).

The protocol for implantation of the nanostructured material involved the following steps: anesthesia and preparation for surgery: the animals were anesthetized by intraperitoneal injection of a mixture of silazine lOmg / Kgc and ketamine 80-100 mg / Kgc ensuring that limb reflexes are suppressed . The mouse was placed in the position for the experiment and then the region where the incision was to be made was prepared: the hair was cut with a hair clipper, epilating cream was applied for about 3 minutes, and after removing it the skin was disinfected with 70% alcohol. A double excision was made at the dorsal part of the mouse using an 8mm diameter preducus (figure 12), which extends to the paniculosus camosus muscle.

SANIMED INTERNATIONAL IMPEX SRL The University of Bucharest Polytechnic University of Bucharest Institute of Virology Ștefan S. Nicolau, Bucharest Catalin-Robertino Prof. Dr. Mircea Prof. Mihnea Dr. Carmen Cristina HEDEG Dumitru Costoiu Diaconu

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11/29/2018

To evaluate the antibacterial efficacy, a 24 μΐ inoculum of bacterial culture (Staphylococcus aureus) of 24 hours density IMcFarland was administered at the level of each excision. After the adsorption period of the inoculum, at the level of each excision the film of collagenic material with Ag was applied which was sewn with resorbable thread in points (figures 12, 13). After surgery, the animals were kept in individual cages, heated and monitored daily for various clinical signs, weight loss and wound closure (Figure 14).

Sampling was performed 4 days after the operation, when the mice were sacrificed by cervical dislocation, after anesthesia with carbon dioxide (CO2). An incision was made around the wound and tissue was taken for molecular biology analyzes with rapid freezing at 80 ° C). Total RNA was extracted with Trizol reagent and reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit with RNAse inhibitor according to the protocols presented above. The qPCR reaction was performed using SyBr Green and specific primers.

The obtained results demonstrate that the inflammatory response induced by the bacterial infection (mIL-1 level) is much diminished in the presence of AgNCh, NaBFU + collagen. A weaker decrease in the IL-1-mediated pro-inflammatory response was also observed in the case of AgNCh material, collagen + NaBH4 (figure 15). It is worth noting the very intense anti-inflammatory effect exerted by the material AgNCh, NaBEU + collagen in the absence of infection, which demonstrates its usefulness in the management of certain types of chronic wounds, characterized by excessive prolongation of the inflammatory phase (Figure 15). Histopathological analysis (hematoxylin-eosin staining) confirmed the existence of a reduced inflammatory process in the case of the AgNCh, collagen + NaBH4 sample (figure 16).

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The University of Bucharest

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Prof. Mihnea Dr. Carmen Cristina

Claims (3)

1. Process for the production of collagen-based antimicrobial hydrogels and silver nanoparticles characterized by the reduction of Ag ions directly in situ in the collagen gel in the presence of reducing agents (eg NaBEL, citric acid) , followed by crosslinking of the hydrogel in the presence of a crosslinking agent (eg glutaraldehyde). Product obtained according to claim 1, characterized in that it is composed mainly of collagen material and Ag nanoparticles (the process allows the variation of the content of Ag nanoparticles in the range of 0.1 - 5%, depending on the biological activity pursued). Depending on the drying method and the concentration of the collagen gel, the collagen matrix obtained may have different degrees of porosity. The product obtained according to claim 2, characterized in that it is composed mainly of collagen material and Ag nanoparticles (the process allows the variation of the content of Ag nanoparticles in the range of 0.1 - 5%, shows antimicrobial activity against Gram-positive bacteria and Gram- negative in suspension and adhered and immunomodulatory activity, by stimulating the installation of the acute inflammatory phase with a role in healing and preventing its prolongation / excessive activation.